Genetic Correlation with the DNA Repair Assay in Mice Exposed to High-LET

We hypothesize that DNA damage induced by high local energy deposition, occurring when cells are traversed by high-LET (Linear Energy Transfer) particles, can be experimentally modeled by exposing cells to high doses of low-LET. In this work, we validate such hypothesis by characterizing and correlating the time dependence of 53BP1 radiation-induced foci (RIF) for various doses and LET across 72 primary skin fibroblast from mice. This genetically diverse population allows us to understand how genetic may modulate the dose and LET relationship. The cohort was made on average from 3 males and 3 females belonging to 15 different strains of mice with various genetic backgrounds, including the collaborative cross (CC) genetic model (10 strains) and 5 reference mice strains. Cells were exposed to two fluences of three HZE (High Atomic Energy) particles (Si 350 megaelectronvolts per nucleon, Ar 350 megaelectronvolts per nucleon and Fe 600 megaelectronvolts per nucleon) and to 0.1, 1 and 4 grays from a 160 kilovolt X-ray. Individual radiation sensitivity was investigated by high throughput measurements of DNA repair kinetics for different doses of each radiation type. The 53BP1 RIF dose response to high-LET particles showed a linear dependency that matched the expected number of tracks per cell, clearly illustrating the fact that close-by DNA double strand breaks along tracks cluster within one single RIF. By comparing the slope of the high-LET dose curve to the expected number of tracks per cell we computed the number of remaining unrepaired tracks as a function of time post-irradiation. Results show that the percentage of unrepaired track over a 48 hours follow-up is higher as the LET increases across all strains. We also observe a strong correlation between the high dose repair kinetics following exposure to 160 kilovolts X-ray and the repair kinetics of high-LET tracks, with higher correlation with higher LET. At the in-vivo level for the 10-CC strains, we observe that drops in the number of T-cells and B-cells found in the blood of mice 24 hours after exposure to 0.1 gray of 320 kilovolts X-ray correlate well with slower DNA repair kinetics in skin cells exposed to X-ray. Overall, our results suggest that repair kinetics found in skin is a surrogate marker for in-vivo radiation sensitivity in other tissue, such as blood cells, and that such response is modulated by genetic variability

53BP1 Repair Kinetics for Prediction of In Vivo Radiation Susceptibility in 15 Mouse Strains

International audienceWe present a novel mathematical formalism to predict the kinetics of DNA damage repair after exposure to both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe). Our method is based on monitoring DNA damage repair protein 53BP1 that forms radiation-induced foci (RIF) at locations of DNA double-strand breaks (DSB) in the nucleus and comparing its expression in primary skin fibroblasts isolated from 15 mice strains. We previously reported strong evidence for clustering of nearby DSB into single repair units as opposed to the classic “contact-first” model where DSB are considered immobile. Here we apply this clustering model to evaluate the number of remaining RIF over time. We also show that the newly introduced kinetic metrics can be used as surrogate biomarkers for in vivo radiation toxicity, with potential applications in radiotherapy and human space exploration. In particular, we observed an association between the characteristic time constant of RIF repair measured in vitro and survival levels of immune cells collected from irradiated mice. Moreover, the speed of DNA damage repair correlated not only with radiation-induced cellular survival in vivo, but also with spontaneous cancer incidence data collected from the Mouse Tumor Biology database, suggesting a relationship between the efficiency of DSB repair after irradiation and cancer risk

Radiation-induced endothelial cells senescence : Molecular pathways and involvement in glioblastoma relapse after radiotherapy

Les dysfonctions endothéliales jouent un rôle important dans la réponse de la tumeur à la radiothérapie. Le glioblastome (GBM), tumeur du système nerveux central, récidive dans 90% des cas dans le champ d’irradiation initial où ont été observées de nombreuses cellules endothéliales en senescence. Lors de ma thèse, j’ai appréhendé les voies moléculaires engageant la sénescence radioinduite des cellules endothéliales et étudié leur implication dans la réponse des cellules de GBM à la radiothérapie. L’irradiation des cellules endothéliales microvasculaires quiescentes, induit la sénescence selon 2 voies moléculaires indépendantes impliquant soit l’antioncogène p53 soit le dysfonctionnement de la chaine respiratoire mitochondriale provoquant la génération d’anion superoxyde. L’inhibition pharmacologique de l’une ou l’autre de ces voies permet de prévenir cette sénescence. Le sécrétome des cellules endothéliales sénescentes (SASP) augmente l’instabilité génomique après irradiation, caractérisée par l’augmentation du nombre de micronoyaux et de division anormale, et la radiorésistance des cellules de GBM. De plus, dans un modèle murin orthotopique, les cellules de GBM irradiées en présence du SASP sont beaucoup plus agressives. La caractérisation du SASP combinée à des études fonctionnelles nous a permis d’identifier le rôle de l’axe CXCL8/CXCL5/CXCR2 dans ces phénotypes. Ces résultats soulignent l’impact de la radiothérapie sur les tissus sains péritumoraux et ses conséquences à long terme sur la résistance tumorale. Le ciblage des potentielles cibles identifiées permettrait d’améliorer le pronostic des GBM.Radiotherapy is one of the main standard cancer treatment. Adjacent tissues, in particular endothelial cells, are never completely spared, especially in the case of highly invasive tumors such as glioblastoma (GBM). This aggressive tumor always relapses in the initial radiation field and the presence of senescent endothelial cells have been shown at this site. The objectives of my thesis were to better characterize the molecular pathways involved in radiationinduced senescence of endothelial cells and to investigate how it might impact GBM responses to radiotherapy. First, using a new model of radiation-induced senescence from primary and quiescent microvascular endothelial cells, we identified two distinct molecular pathways involved in long-term senescence: the canonic pathway p53 and an undescribed mitochondrial pathway involving respiratory chain dysfunction and chronic superoxide anion production. Pharmacological inhibition of either one of these pathways prevents endothelial cell senescence. Then, using biochemical and phenotypic analyses, we unrevealed a crucial role of their secretome in radiation response of GBM cells. Indeed, secreted CXCL8 and CXCL5 by senescent endothelial cells increase both genomic instabilities, displayed by polynucleidy, abnormal division and micronuclei formation, and radio-resistance of tumor cells, leading ultimately to more aggressive tumors in a murine orthotopic GBM model. These results highlight the impact of irradiation on healthy tissues and its potential impact on tumor reccurence. Targeting the molecular actors identified during my thesis, could reduce GBM relapse and increase patient survival

Targeting PRMT5 enhances the radiosensitivity of tumor cells grown in vitro and in vivo

Abstract PRMT5 is a widely expressed arginine methyltransferase that regulates processes involved in tumor cell proliferation and survival. In the study described here, we investigated whether PRMT5 provides a target for tumor radiosensitization. Knockdown of PRMT5 using siRNA enhanced the radiosensitivity of a panel of cell lines corresponding to tumor types typically treated with radiotherapy. To extend these studies to an experimental therapeutic setting, the PRMT5 inhibitor LLY-283 was used. Exposure of the tumor cell lines to LLY-283 decreased PRMT5 activity and enhanced their radiosensitivity. This increase in radiosensitivity was accompanied by an inhibition of DNA double-strand break repair as determined by γH2AX foci and neutral comet analyses. For a normal fibroblast cell line, although LLY-283 reduced PRMT5 activity, it had no effect on their radiosensitivity. Transcriptome analysis of U251 cells showed that LLY-283 treatment reduced the expression of genes and altered the mRNA splicing pattern of genes involved in the DNA damage response. Subcutaneous xenografts were then used to evaluate the in vivo response to LLY-283 and radiation. Treatment of mice with LLY-283 decreased tumor PRMT5 activity and significantly enhanced the radiation-induced growth delay. These results suggest that PRMT5 is a tumor selective target for radiosensitization

Ionizing radiation induces long-term senescence in endothelial cells through mitochondrial respiratory complex II dysfunction and superoxide generation

International audienceIonizing radiation causes oxidative stress, leading to acute and late cellular responses. We previously demonstrated that irradiation of non-proliferating endothelial cells, as observed in normal tissues, induces early apoptosis, which can be inhibited by pretreatment with Sphingosine-1-Phosphate. We now propose to better characterize the long-term radiation response of endothelial cells by studying the molecular pathways associated with senescence and its link with acute apoptosis. First, senescence was validated in irradiated quiescent microvascular HMVEC-L in a dose- and time-dependent manner by SA β-galactosidase staining, p16Ink4a and p21Waf1 expression, pro-inflammatory IL-8 secretion and DNA damage responseactivation. This premature aging was induced independently of Sphingosine 1-Phosphate treatment, supporting its non-connection with acute IR-induced apoptosis. Then, senescence under these conditions showed persistent activation of p53 pathway and mitochondrial dysfunctions, characterized by O2●- generation, inhibition of respiratory complex II activity andover-expression of SOD2 and GPX1 detoxification enzymes. Senescence was significantly inhibited by treatment with pifithrin–α, a p53 inhibitor, or by MnTBAP, a superoxide dismutase mimetic, validating those molecular actors in IR-induced endothelial cell aging. However, MnTBAP, but not pifithrin–α, was able to limit superoxide generation and to rescue the respiratory complex II activity. Furthermore, MnTBAP was not modulating p53 up-regulation, suggesting that IR-induced senescence in quiescent endothelial cells is provided by at least 2 different pathways dependent of the mitochondrial oxidative stress response and the p53 activation. Further characterization of the actors involved in the respiratory complex II dysfunction will open new pharmacological strategies to modulate late radiation toxicity

53BP1 Repair Kinetics for Prediction of In Vivo Radiation Susceptibility in 15 Mouse Strains.

We present a novel mathematical formalism to predict the kinetics of DNA damage repair after exposure to both low- and high-LET radiation (X rays; 350 MeV/n 40Ar; 600 MeV/n 56Fe). Our method is based on monitoring DNA damage repair protein 53BP1 that forms radiation-induced foci (RIF) at locations of DNA double-strand breaks (DSB) in the nucleus and comparing its expression in primary skin fibroblasts isolated from 15 mice strains. We previously reported strong evidence for clustering of nearby DSB into single repair units as opposed to the classic "contact-first" model where DSB are considered immobile. Here we apply this clustering model to evaluate the number of remaining RIF over time. We also show that the newly introduced kinetic metrics can be used as surrogate biomarkers for in vivo radiation toxicity, with potential applications in radiotherapy and human space exploration. In particular, we observed an association between the characteristic time constant of RIF repair measured in vitro and survival levels of immune cells collected from irradiated mice. Moreover, the speed of DNA damage repair correlated not only with radiation-induced cellular survival in vivo, but also with spontaneous cancer incidence data collected from the Mouse Tumor Biology database, suggesting a relationship between the efficiency of DSB repair after irradiation and cancer risk